The detection of replicating cells is usually accomplished by demonstrating that [.sup.3 H]-thymidine, or other DNA precursor, is incorporated into DNA. This is detected by either autoradiography or scintillation counting, as described by Taylor, et al, Proc. Natl. Acad. Sci., U.S.A. 43:122 (1957) and R. Baserga and D. Malamud, Autoradiography: Technique and Application, Harper, N.Y. (1969).
The process of autoradiography involves overlaying a microscope slide having [.sup.3 H]-DNA incorporated on it with a photographic emulsion. The emulsion-bearing slide is stored for a time sufficient to expose the emulsion to particles emitted from the decaying tritium; this period usually takes several days and may last as long as several weeks. The slide is photographically developed revealing "fogging" of the emulsion caused by radiation issued from the decaying tritium. This radiation causes the silver grains in the emulsion to be visible over only those cells which have incorporated [.sup.3 H]-thymidine into their DNA. The proportion of DNA-synthesizing cells in the total population is then enumerated microscopically. Approximate quantitation of the amount of radioactive DNA synthesized per cell can be estimated by counting the silver grains overlying each cell.
Another method of measuring DNA synthesis involves the quantitation of isotopic incorporation of a DNA precursor, such as [.sup.3 H]-thymidine, [.sup.14 C]-thymidine, or [.sup.125 I] iododeoxyuridine, or one of the other nucleosides which are DNA precursors and are readily incorporated into DNA. The DNA of a number of cells is extracted and treated then counted in a liquid scintillation spectrometer.
While autoradiography is used to measure replication in individual cells, scintillation counting of the incorporated radioactivity is a "batch" process in which only the total amount of radioactivity is measured. Thus, the frequency of replicating cells in a population cannot be measured. It is also to be noted that both autoradiography and scintillation counting techniques require the use of radioisotopes; this contributes a radiation hazard, with attendant handling and disposal precautions.
The process of DNA synthesis can also be measured by the use of 5-iododeoxyuridine and 5-bromodeoxyuridine, both of which are chemical analogues of thymidine. Since these compounds appear to the cell as thymidine, they are usually incorporated into DNA in place of thymidine; see Hughes, et al, Fed. Proc. 23:640 (1964). Thus, the de novo synthesis of DNA can be monitored by measuring in some manner the incorporation of these base analogues by isotopic techniques (.sup.125 IdUrd), as described above, or increased DNA density (Lark, et al, Biochim. Biophys. Acta, 76, 9-24 1963), as fluorescence quenching or enhancement of fluorescence of certain DNA-binding dyes, as described by Latt, Proc. Natl. Acad. Sci. (U.S.A.) 70:3395 (1973); and Swartzendruber, Exp. Cell. Res. 109:439 (1977).
Antibodies specific for BrdUrd and IdUrd were produced in rabbits by Sawicki, et al, Science 174:70 (1977). In this study rabbits were immunized with bovine serum albumin conjugates of 5-bromouracil, 5-iodobromouracil, and 6-methyladenosine which produced antibodies specific for the bases. These antibodies were used to detect immunochemically 5-bromouracil and 6-methyladenosine in denatured DNA. This type of antibody was applied to the detection of DNA replication in mammalian cells by immunocytochemical means, see Gratzner, et al, Exp. Cell Res. 95:88 (1975) and J. Histochem. Cytochem. 24:34 (1976); and by flow cytometry, Gratzner and Leif, Cytometry 1:385 (1981).
While antibodies raised in rabbits are useful, crossreaction of the antibodies with thymidine limited the utility of the immunofluorescent technique, since it was difficult to absorb out all of the crossreacting species; accurate titrations were found to be necessary in order to obtain an optimal concentation of antibody which would react only with bromodeoxyuridine and not with thymidine. The monoclonal antibody of the present invention only reacts with bromodeoxyuridine or iododeoxyuridine; no crossreaction whatever has been observed with thymidine at any concentration of antibody tested. Thus, the monoclonal antibody has the advantage of not requiring titration and thus much greater signal to noise ratio (fluorescence intensity of BrdUrd-incorporated DNA:intensity of control).
The present invention relates to a process for producing in living cells antibodies specific for 5-bromodeoxyuridine and 5-iododeoxyuridine and more specifically to the production of monoclonal antibodies that are specific for those compounds from fused, hybrid cells.
The antibodies produced by such cells either in culture or in vivo can be used to detect cell proliferation in vivo or in vitro. The antibodies can also be used for the isolation of segments of DNA containing these bases or for the immunological detection of replicating DNA by fluorescence, bright field or by electron microscopy.